Browsing by Author "Berget, Susan M."

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    Analysis of HeLa cell premessenger RNA splicing complexes containing the snRNP U1 by native gel electrophoresis
    (1988) Zillmann, Martin; Berget, Susan M.
    The typical eukaryotic RNA polymerase II primary transcript is divided into regions that encode information expressed at the protein level (exons) and those which do not (introns). The latter must be removed from the transcript rapidly and with proper joining of the coding sequences during the maturation of the transcript in the nucleus. This process is termed splicing and is accompanied by the sequential addition of factors to the primary transcript resulting in the formation of a series of large ribonucleoprotein particles. The splicing reaction can be studied in vitro in HeLa cell nuclear extracts by the addition of a capped, in vitro transcribed splicing precursor RNA. A native gel electrophoresis system was developed which allowed resolution of various ribonucleoprotein complexes and the study of the splicing complex and intermediates in its formation. The HeLa cell nuclear extracts were found to immediately assemble exogenously added precursor RNAs into rapidly migrating complexes. With time, complexes migrating more slowly were observed. The complex migrating the most slowly appeared concurrently with the products of 5$\sp\prime$ junction cleavage. This complex was identified as the "active" splicing complex by the presence of reaction intermediates and the requirement for both ATP and splicing consensus sequences for its formation. Later in the reation, when large amounts of ligated RNA had been generated, rapidly migrating complexes reappeared. All of these complexes contained U snRNPs as defined by immunoprecipitation of gel-fractionated complexes. In particular, eluted active complex contained U1 snRNPs as defined by the ability of anti-U1 antiserum to immunoprecipitate this particle in the presence of competing free, unlabelled precursor RNA. Previously reported gel systems appear to resolve active complexes devoid of U1 snRNPs, a snRNP known to be required for splicing both in vivo and in vitro. Furthermore, the 5$\sp\prime$ splice junction, the region to which U1 snRNPs bind, was protected from oligonucleotide directed RNase H cleavage in eluted complexes, indicating that a 5$\sp\prime$ factor remained bound during electrophoresis. Apparently, complexes eluted from the native system retain most of the properties, other than activity, found for these complexes in whole extracts, suggesting that this gel system is an ideal tool for the study of the ribonucleoprotein complexes involved in splicing.
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    Biochemical and genetic characterization of in vitro pre-messenger-RNA polyadenylation
    (1987) Sperry, Ann O'Brien; Berget, Susan M.
    The sequence and biochemical requirements of pre-mRNA polyadenylation were investigated using the simian virus 40 (SV40) early polyadenylatin site. This signal directs 3$\sp\prime$ processing of two mRNA molecules coding for the large and small T-antigens of the DNA tumor virus SV40. The processing of this site was studied using a nuclear extract capable of accurately and efficiently polyadenylating exogenously provided RNA molecules. The sequence requirements of polyadenylation were examined by point mutagenesis of conserved sequence elements within the SV40 early site followed by assay of their effect on in vitro polyadenylation. Alteration of the internal pyrimidine of the highly conserved sequence AAUAAA from U to an A abolishes cleavage at the normal polyadenylation site and activates a minor site downstream of the distal, usually inactive, hexanucleotide. Point mutation of the conserved CAYUG element decreases the efficiency of cleavage without changing its specificity. Complete removal of this sequence abolishes cleavage of the SV40 early polyadenylation site. These results suggest that multiple sequence elements are involved in generating accurately polyadenylated RNA from the SV40 early genes. The ATP requirement of the polyadenylation cleavage reaction was examined by removal of ATP from in vitro reactions using the SV40 early and the adenovirus L3 substrate RNAs. In vitro polyadenylation of the SV40 early site requires 1mM ATP to meet the energy requirements of endonucleolytic cleavage as well as to provide substrate for polymerization. At ATP concentrations below 0.1 mM, the SV40 early polyadenylation site is subject to alternate cleavage approximately 21 nucleotides downstream of the correct cleavage site. Adenovirus L3 RNA is not an efficient substrate for alternate cleavage in the absence of ATP. This alternate activity has many characteristics in common with the correct cleavage. The behavior of the ATP-independent cleavage product is inconsistent with its being an intermediate in the polyadenylation reaction. The ATP-independent cleavage activity requires an intact polyadenylation consensus signal, a 3$\sp\prime$ terminus located within 100 nucleotides of the polyadenylation site, and extract U-snRNPs. These requirements suggest that ATP-independent cleavage is an alternate activity of the normal polyadenylation machinery observations suggest that SV40 early and adenovirus L3 RNAs are differentially recognized by the polyadenylation machinery. (Abstract shortened with permission of author.)
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    Classes of polyadenylation sites revealed by native gel electrophoresis of in vitro assembled complexes and sensitivity to U RNA cleavage
    (1988) Rose, Scott Daniel; Berget, Susan M.
    The sequences that comprise a polyadenylation signal are varied. With the exception of the conserved hexanucleotide AAUAAA, other required sequence elements vary from site to site. This variability in sequence content may be indicative of different types or classes of polyadenylation signals. We have used an in vitro polyadenylation system to investigate the possibility that classes of poly(A) sites exist. Precursor RNAs from the SV40 late and adenovirus 2 L3 polyadenylation sites were examined for differences in: assembly into RNA-protein complexes; sequence requirements for complex assembly; and interactions with small nuclear ribonucleoproteins (snRNPs). Both SV40 late and L3 precursor RNA required an intact hexanucleotide and downstream sequence elements for complex formation. The stability of the complexes assembled using the two precursor RNAs was different. The L3 RNA complex was unstable in the presence of the anion poly(ACU); whereas the SV40 late complex or a chimeric L3/SV40 late complex were not. SV40 late and L3 precursor RNAs associated with the Sm protein determinant (common to U1, U2, U4/U6, U5 and U7 snRNPs) and a U1 snRNP-specific protein determinant early in the polyadenylation reaction. This association was reduced as the polyadenylation reaction progressed. Formation of the polyadenylation specific complexes was shown to require the small nuclear RNAs (snRNAs) U1, U2 and U4. The two polyadenylation precursor RNAs showed a different sensitivity to oligonucleotide directed RNase H cleavage of U RNAs. The SV40 late site was sensitive to cleavages of U1, U2 and U4 RNA. The L3 site showed sensitivity to only U4 cleavage. When the two polyadenylation signals were preceded by a functional intron with 5$\sp\prime$ and 3$\sp\prime$ splice sites, sensitivity to U RNA cleavages was altered. However even as chimeric polyadenylation splicing templates, the two sites exhibited different sensitivities to U4 cleavage in the region of U4 in which it hybridizes to U6 RNA. The observed differences in complex stability, and sensitivity to U RNA cleavage suggest that different classes of polyadenylation sites exist.
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    Construction of recombinant molecules containing RNA processing signals and binding of human snRNPs to some nucleic acids
    (1984) Nees, David; Berget, Susan M.; Palmer, Graham; Bennett, George
    The restriction endonuclease Pst I cleavage site map of Ad2 was generated. Several recombinant molecules were formed, containing Pst I-generated fragments of Ad2 DNA, inserted into the E. coli vector pBR322. These molecules are designed to be useful for the study of RNA processing because they contain signals for splicing and polyadenylation. A filter binding assay was developed to measure the affinity of snRNPs for nucleic acids. The of U1 snRNPs was determined to be on the order of 1 to 11 M for RNA with or without a donor splice junction and for denatured DNA. U2 snRNP also bound RNA containing a donor splice junction with a KD of 1 M. The dissociation of U1 snRNP from denatured DNA was followed and the remainder of the snRNPs dissociated in less than one minute and fifty percent dissociated with a t1/2 of 2 minutes.
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    Isolation and characterization of human small nuclear ribonucleoproteins
    (1984) Kinlaw, Claire S.; Berget, Susan M.; Matthews, Kathleen S.; Beckingham, Kathleen M.; Bennett, George; Kellems, Rodney E.
    Human small nuclear ribonucleoproteins (snRNPs) containing U snRNAs have been fractionated into three RNA-specific populations, and snRNPs containing U1 and U2 snRNAs have been isolated~by biochemical methods. U1 and U2 snRNPs remained immunoprecipitable by Systemic Lupus Erythematosus antibodies during isolation, and purified snRNPs contained polypeptides of the same molecular weights as those defined by immunoprécipitation of crude extracts. The polypeptide components of U1 and U2 snRNPs have been compared by two-dimensional gel electrophoresis and immunobinding. U1 and U2 snRNPs contained both unique and common polypeptides. Purified U1 snRNPs contained U1 RNA and 1 snRNP polypeptides of molecular weights 67, (P67), 3, (P3), 23, (P23F), 21,5 (P22), 17,5 (P18), 2 at 12,3 (P12F and P12S), 1,2 (P1), 9,1 (P9), and 8,5 (P8). Purified U2 snRNPs contained U2 RNA and 9 snRNP polypeptides including the common polypeptides P23F, P22, P12F, P12S, P1, P9, and P8 as well as two U2 specific polypeptides of 23, (P23S) and 27, (P27) daltons. Five of the common polypeptides, including P23F, P22, P12F, P12S, and P9, were basic and may be important in snRNP assembly. One common polypeptide, P1 was acidic, and the remaining common polypeptide, P8, was neutral. Two of the U1 specific polypeptides, P3 and P18, as well as the two U2 specific polypeptides, P27 and P23S, were neutral. The third Ul specific polypeptide, P67, was slightly basic. Two of the common polypeptides, P23F and P22, were present in nonstoichiometric amounts and were recognized by a monoclonal anti-Sm antibody. Only one of them is present in rodent cells (Conner et al., 1982). Thus, in human cells, P23F and P22 may be two variants of the same polypeptide, with each snRNP complex containing either P23F or P22. P12F is also recognized by the same monoclonal antibody, suggesting it may be related to P23F and P22. Because Ul and U2 snRNPs contain common and unique polypeptides, it is suggested that they serve similar but distinct functions in. vivo. Ul has been implicated in hnRNA splicing (Lerner et al., 198; Rogers and Wall, 198). U2 may be involved in another aspect of hnRNA processing.
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    The effect of an oral or intravenous nucleotide-free diet on selected enzyme activities in purine metabolism in rats
    (1982) Snyder, Sandra Lynn; Rudolph, Frederick B.; Berget, Susan M.; Schroepfer, George J.; Bennett, George; Storck, Roger L.
    Interrelationships between purine metabolism and immunity, cancer, and the diet have been considered. In studying trends of metabolic changes which occur in response to changes in the purine content of the diet, it has been hypothesized that when purines are lacking from the diet, there is a general shift from a catabolic to an anabolic state. In the present investigation, the activities of selected enzymes on purine metabolism in rats were studied with respect to an oral or intravenous nucleotide-free diet compared to the activities in rats fed normal chow. The intravenous nucleotide-free diet caused a decrease in purine nucleoside phosphorylase activity, an increase in adenine phosphoribosyl transferase, and no change in the activity of hypoxanthine-guanine phosphoribosyl transferase, with respect to a normal control diet. The orally fed nucleotide-free diet caused a decrease in the activity of purine nucleoside phosphorylase and xanthine oxidase and increase in pancreatic ribonucléase and no change in adenine phosphoribosyl transferase or hyphoxanthine-guanine phosphoribosyl transferase, with respect to a normal control diet. These observations support the predicted shift from catabolism to anabolism. They also augment the growing awareness of the interrelationships between diet and cancer as well as diet and the immune response.